The invention is directed toward methodologies and apparatus for use in the preparation of acellular, i.e. essentially lacking in living cells and/or non-living cells, soft-tissue implants, in small quantities and commercializable quantities. Such soft-tissue implants include vascular graft substitutes. These implants can be derived from tissue engineered soft tissue devices, tissue products derived from animal or human donors that contain or are devoid of cells, and that contain or are devoid of valve structures useful in directing the flow of fluids through tubular vascular devices, and/or combinations of natural tissue products and tissue engineered soft-tissue products. The invention includes methodologies and apparatus for producing uniform, gently processed, decellularized multiple soft tissue implants, where processing time is significantly reduced and the number of implants produced per day is increased. The decellularized grafts produced are significantly improved in long-term durability and function when used in clinical applications.
Numerous types of vascular graft substitutes have been produced in the last four decades. These vascular graft substitutes have included large and small diameter vascular, blood carrying tubular structures, grafts containing valvular structures (vein substitutes, and heart valve substitutes) and lacking valvular structures (artery substitutes). The materials out of which these vascular grafts have been constructed have included man-made polymers, notably Dacron and Teflon in both knitted and woven configurations, and non-man-made polymers, notably tissue engineered blood vessels such as described in U.S. Pat. Nos. 4,539,716; 4,546,500; 4,835,102; and blood vessels derived from animal or human donors such as described in U.S. Pat. Nos. 4,776,853; 5,558,875; 5,855,617; 5,843,181; and 5,843,180.
The prior art processing methods are prohibitively time consuming, easily requiring numerous days, for example anywhere from eight to twenty-one days total processing time. Such long processing times result in proteolytic degradation of the matrix structures of the processed tissues. Over the past few decades numerous efforts have been made to manage the large surgical use of vascular prostheses in the treatment of vascular dysfunctions/pathologies. While vascular prostheses are available for clinical use, they have met with limited success due to cellular and immunological complications, and the inability to remain patent and function. These problems are especially pronounced for small diameter prostheses, i.e. less than about 6 mm. Efforts have been directed at removing those aspects of allograft and xenograft vascular prostheses that contribute to immunological xe2x80x9crejectionxe2x80x9d and these efforts have focused primarily on development of various xe2x80x9cdecellularizationxe2x80x9d processes, which processes require unduly burdensome incubation times. In addition the prior art methods involve using volumes of processing solutions which do not lend themselves to the production of large numbers of vascular grafts, which ability to xe2x80x9cscale-upxe2x80x9d is necessary for economic clinical use.
The inventive process produces acellular grafts including but not limited to ligaments, tendons, menisci, cartilage, skin, pericardium, dura mater, fascia, small and large intestine, placenta, veins, arteries, and heart valves. The process is advantageous over prior art processes in that processing times and conditions have been optimized and reduced, and the economics of production have been dramatically improved, resulting in large numbers of uniform, non-immunogenic grafts being produced. The grafts produced are non-immunogenic, are substantially free from damage to the matrix, and are substantially free from contamination including for example free from infectious agents.
The invention involves the use of an anionic agent, for example sodium dodecylsulfate (SDS), for the treatment of tissues with the dual objective of decellularization and treatment of tissues to restrict recellularization. Further, the invention expands on the process of treating tissue(s) with SDS, describing how the amount(s) of SDS deposited in the tissue(s) can be further enhanced/reduced to either further inhibit recellularization of the tissue OR enhance recellularization of the tissue. Treatment of tissues with salt solutions prior to treatment with SDS results in different patterns of SDS deposition/precipitation in the tissues than treatment of tissues with SDS followed by treatment of tissues with salt solutions. Treatment of tissues with SDS prior to salt treatment can be expected to result in significant binding of SDS, primarily via hydrophobic interactions, to matrix xe2x80x9cproteinsxe2x80x9d with further deposition of SDS in the tissues as salt precipitated materials by salt precipitation post SDS treatment. Treatment of tissues with salt solutions prior to treatment with SDS solutions can be expected to result in significant precipitation of SDS as a salt precipitated form and less SDS being bound to tissue matrix structure(s) via hydrophobic interactions. It is further understood that the particular salt solution used, either prior to or following SDS treatment, can significantly alter the subsequent solubility of the salt precipitated SDS and thus long-term retention of SDS in the tissues post implantation. The observed salt effects on both precipitability of SDS and subsequent resolubilization of the salt precipitated form of SDS indicate an activity order of Ca greater than Mg greater than Mn greater than K greater than Na and calcium salts of dodecylsulfate (CaDS) are less soluble and thus more slowly released from treated tissues than, for example, sodium salts of dodecylsulfate (SDS). The invention is directed at a process for producing acellular soft-tissue implants including vascular grafts, veins, arteries, and heart valves, where processing times and conditions have been optimized to dramatically improve on the economics of production as well as to produce a graft with minimum damage to the matrix structure of the acellular graft. It is a further objective of the present invention to describe how to control the amount(s) of anionic detergents, for example sodium dodecylsulfate (SDS), deposited in the tissue(s) with the objective of enhancing or restricting subsequent recellularization.
The inventive process is a process for preparing biological material(s) for implantation into a mammalian cardiovascular system, musculoskeletal system, or soft tissue system. The process removes cellular membranes, nucleic acids, lipids, and cytoplasmic components and produces an implant having an extracellular matrix including as major components collagens, elastins, proteoglycans, and mucopolysaccharides.
The process provides for the production of commercializable quantities of acellular soft tissue grafts for implantation into mammalian systems by removing the cellular populations, cellular remnants, nucleic acids, and small molecular weight proteins, lipids, and polysaccharides forming an acellular nonsoluble matrix having as major components collagens, elastins, hyaluronins, and proteoglycans. The acellular tissue produced can be implanted into a mammalian system, or recellularized in vitro and subsequently implanted into a mammalian system.
An embodiment of the process includes the following steps:
isolating from a suitable donor a desired tissue sample of the biological material;
extracting the tissue with mildly alkaline hypotonic buffered solution of an endonuclease such as Benzonase(copyright) (a registered product of Merck KGaA, Darmstadt, Germany) and a nonionic detergent formulation such as Allowash Solution(trademark) (a registered trademark product of LifeNet, Virginia Beach, Va.);
optionally treating the tissue with a hypertonic buffered salt solution;
extracting and treating the tissue with a mildly alkaline hypotonic buffered solution of sodium dodecylsulfate, optionally with 0.1 to 0.5 M sodium chloride rendering the solution hypertonic;
optionally treating the tissue with a hypertonic buffered salt solution;
washing the tissue with ultrapure water followed by a water solution of chlorine dioxide; and
storage in a sealed container in isotonic saline, chlorine dioxide or 70% isopropanol.
The invention provides a process for preparing an acellular soft tissue graft for implantation into a mammalian system, including extracting a soft tissue sample with an extracting solution including one or more nonionic detergents and one or more endonucleases, to produce extracted tissue; treating the extracted tissue with a treating solution including one or more anionic detergents, to produce a treated tissue; washing the treated tissue with a decontaminating solution including one or more decontaminating agents to produce the acellular soft tissue graft; and storing the acellular soft tissue graft in a storage solution comprising one or more decontaminating agents.
The invention further provides a process for preparing commercializable quantities of acellular soft tissue grafts for implantation into mammalian systems, including obtaining tissue samples from an acceptable donor; extracting the tissue samples with an extracting solution including one or more nonionic detergents and one or more endonucleases, to produce extracted tissue; treating the extracted tissue with a treating solution including one or more anionic detergents, to produce a treated tissue; washing the treated tissue with a decontaminating solution including one or more decontaminating agents to produce the acellular soft tissue graft; and storing the acellular soft tissue graft in a storage solution including one or more decontaminating agents.
The invention also provides a process for preparing an acellular soft tissue graft for implantation into a mammalian system, including inducing a pressure mediated flow of an extracting solution including one or more nonionic detergents and one or more endonucleases, through soft tissue, to produce extracted tissue; inducing a pressure mediated flow of a treating solution including one or more anionic detergents, through the extracted tissue, to produce a treated tissue; inducing a pressure mediated flow of a decontaminating solution including one or more decontaminating agents through the treated tissue, to produce the acellular soft tissue graft; and storing the acellular soft tissue graft in a storage solution including one or more decontaminating agents.
The invention provides a process where the extracting solution is recirculated through the soft tissue graft.
The invention further provides a process where the treating solution is recirculated through the soft tissue graft.
The invention also provides a process where the decontaminating solution is recirculated through the soft tissue graft.
The invention provides a process for producing an acellular tissue graft and includes the use of calcium salts which use results in the acellular tissue graft containing a significantly more insoluble form of salt precipitated anionic detergent, which results in retarded recellularization of the acellular tissue graft in vivo or in vitro.